The recent observations that both DNA adducts and oxidative base damage are increased in the brains from Alzheimer s and Parkinson s patients and in spinal cord tissue of patients with amyotrophic lateral sclerosis (ALS) support the idea that oxidative DNA damage may contribute to the observed loss of neurons in these neurological disorders (8,9). Therefore, understanding how oxidative DNA damage is repaired (or not) is critical for understanding the pathology underlying these diseases. The overall hypothesis of this proposal is that oxidative stress induced DNA damage in neuronal cell lines leading to cellular malfunction and/or death and DNA repair enzymes are critical for the protection of neurons against oxidative stress. The following specific Aims will address this hypothesis.
Specific Aim 1 : Functional and biochemical characterization of the repair enzymes Ogg1, NTH, MTH, and MYH in the neuroblastoma cell lines SHSY5Y and Neuro-2A and in primary cultures of hippocampal neurons. We will measure the steady- state level of mRNA of the four repair enzymes, cytotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG in the absence and presence of the following agents: L-Dopa, dopamine, menadione, and H2O2. Experiments will also be performed in post-mitotic SHSY5Y cells.
Specific Aim 2 : Determine the effects of over-expressing these oxidative DNA repair genes on extotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG following exposure to L-Dopa, dopamine, menadione, or H2O2. Both mitotic and post-mitotic SHSY5Y cells and the Neuro-2A cells will be analyzed.
Specific Aim 3 : Determine the effects of antisence gene expression specific for the four oxidative DNA repair genes on cytotoxocity, apoptosis, frequency of mutations, and the amount of 8-oxoG/FapyG in the absence and presence of the following agents: L-Dopa, dopamine, menadione, and H2O2 in SHSY5Y mitotic and post-mitotic cells. If increasing the levels of DNA repair enzymes protects cells against oxidative stress, then this could provide a novel therapeutic basis for the treatment of neurological disorders such as ALS, aging, Alzheimer s and Parkinson s diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS038506-04
Application #
6629310
Study Section
Special Emphasis Panel (ZRG1-MDCN-2 (01))
Program Officer
Oliver, Eugene J
Project Start
2000-02-14
Project End
2005-01-31
Budget Start
2003-02-01
Budget End
2005-01-31
Support Year
4
Fiscal Year
2003
Total Cost
$324,200
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
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Vasko, Michael R; Guo, Chunlu; Kelley, Mark R (2005) The multifunctional DNA repair/redox enzyme Ape1/Ref-1 promotes survival of neurons after oxidative stress. DNA Repair (Amst) 4:367-79
Tell, Gianluca; Damante, Giuseppe; Caldwell, David et al. (2005) The intracellular localization of APE1/Ref-1: more than a passive phenomenon? Antioxid Redox Signal 7:367-84
Rinne, Mikael; Caldwell, David; Kelley, Mark R (2004) Transient adenoviral N-methylpurine DNA glycosylase overexpression imparts chemotherapeutic sensitivity to human breast cancer cells. Mol Cancer Ther 3:955-67
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Wang, Dong; Luo, Meihua; Kelley, Mark R (2004) Human apurinic endonuclease 1 (APE1) expression and prognostic significance in osteosarcoma: enhanced sensitivity of osteosarcoma to DNA damaging agents using silencing RNA APE1 expression inhibition. Mol Cancer Ther 3:679-86
Kremer, Ted M; Rinne, Mikael L; Xu, Yi et al. (2004) Protection of pulmonary epithelial cells from oxidative stress by hMYH adenine glycosylase. Respir Res 5:16
Fishel, Melissa L; Seo, Young R; Smith, Martin L et al. (2003) Imbalancing the DNA base excision repair pathway in the mitochondria; targeting and overexpressing N-methylpurine DNA glycosylase in mitochondria leads to enhanced cell killing. Cancer Res 63:608-15
Ladd, Paula D; Wilson 3rd, David M; Kelley, Mark R et al. (2003) Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity. DNA Repair (Amst) 2:187-98
Roth, Timothy J; Xu, Yi; Luo, Meihua et al. (2003) Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection. Cancer Gene Ther 10:603-10

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